Streptococcus is a genus of spherical shaped Gram-positive bacteria. Clinically, individual species of streptococci are classified primarily based on their Lancefield serotyping—according to specific carbohydrates in the bacterial cell wall. These are named Lancefield groups A to T. However the pathogens in these different groups share many similarities at the genetic level. For example Streptococcus equi (which is in group C1 and which is the causative agent of equine strangles) shares 80% genome identity with the human pathogen S. pyogenes (which is in group A, and which is the causative agent of many human conditions including strep throat, acute rheumatic fever, scarlet fever, acute glomerulonephritis and necrotizing fasciitis). Additionally the two organisms share many near identical toxins and virulence factors. Streptococci are further characterised via their haemolytic properties. Alpha haemolysis is caused by a reduction of iron in haemoglobin giving it a greenish color on blood agar. Beta only haemolysis is complete rupture of red blood cells giving distinct, wide, clear areas around bacterial colonies on blood agar. Other streptococci are labeled as gamma haemolytic.
Strangles, caused by Streptococcus equi subspecies equi (S. equi), is the most frequently diagnosed infectious disease of horses worldwide and is responsible for significant welfare cost and economic losses to the equine industry. The disease is characterised by abscessation of the lymph nodes of the head and neck. Abscesses formed in the retropharyngeal lymph nodes usually rupture into the guttural pouches, which drain via the nostrils leading to the classical mucopurulent nasal discharge associated with strangles. However, the purulent material in a proportion of guttural pouches fails to drain completely. Over time this becomes inspissated, enabling live S. equi to persist in horses that have recovered from the acute disease for up to several years in the absence of obvious clinical signs (Newton et al., 1997). S. equi periodically sheds from persistently infected carrier horses allowing transmission to naïve individuals and resulting in new outbreaks of disease. The generation and persistence of carriers within equine populations is proposed to have been critical to the global spread of S. equi infection and the efficient identification and treatment of carriers is key to the prevention and eradication of this important disease.
Traditionally the diagnosis of S. equi infection was based upon the culture of this β-haemolytic organism using selective media, followed by biochemical tests, which rely on the inability of S. equi to ferment lactose, sorbitol or ribose (Bannister et al., 1985). The isolation of β-haemolytic colonies is complicated by the presence of other bacteria most notably the closely related opportunistic pathogen Streptococcus equi subspecies zooepidemicus (S. zooepidemicus). This β-haemolytic organism confounds the isolation of S. equi leading to the generation of potential false-negative results. Furthermore, as the culture test identifies S. equi through a lack of sugar fermentation, strains of S. zooepidemicus that also fail to ferment these sugars (Holden et al., 2009) can be mis-identified as S. equi leading to the reporting of false-positive results. Finally, the isolation and identification of S. equi is time consuming and requires a minimum of 48 hours from receipt of clinical samples. This reporting delay often has consequences for the isolation of infected horses providing S. equi with greater opportunity to transmit through the horse population.
Advances in nucleic acid based technologies and the increased number of published pathogen genome sequences has had a significant impact on the diagnosis of infectious diseases. The first PCR-based test developed for S. equi targeted the 5′ region of the SeM gene, which encodes a cell wall anchored protein, thought to be important in the virulence of S. equi. Using this target, up to 3 times more clinical samples were positive for S. equi than by culture and biochemical tests alone (Newton et al., 2000; Timoney and Artiushin, 1997).
Historically, the SeM gene was thought to be non-variant based upon its HindIII restriction pattern on Southern blotting (Galan and Timoney, 1988). However, this has since been disproved in a number of separate studies that have demonstrated that not only is this region of the SeM gene highly variable (Anzai et al., 2005; Kelly et al., 2006), with 99 alleles identified to date (http:/pubmlst.org/perl/mlstdbnet/agdbnet.pl?file=sz_seM.xml, last accessed Nov. 12, 2011), but that it is also frequently deleted in strains of S. equi isolated from persistently infected carriers (Chanter et al., 2000). These results highlight that SeM should no longer be considered a suitable PCR target for the diagnosis of S. equi infection as sequence variation in primer binding sites and loss of the target may lead to the reporting of false-negative results.
The loss of diagnostic PCR targets leading to incorrect reporting can have serious consequences for the control of infectious disease. In Sweden, the occurrence of a 377 bp deletion in CDS1 of the pSW2 plasmid of the human pathogen Chlamydia trachomatis resulted in the false-negative diagnosis of many infected patients, and the rapid spread of this variant within the population, to the extent that in Sweden the variant accounted for 20-64% of current infections (Seth-Smith et al., 2009). The completion of the S. equi strain 4047 (Se4047) and S. zooepidemicus strain H70 (SzH70) genome sequences (Holden et al., 2009) has enabled the identification of alternative S. equi-specific targets suitable for multiplex diagnostic PCR-based tests, that reduce the risk of false-negative reporting.
A multiplex assay based upon a region of the superoxide dismutase gene (sodA) present in both S. equi sub-species and the S. equi-specific gene seeI was developed to identify and differentiate S. equi and S. zooepidemicus isolated on culture plates (Baverud et al., 2007). However, the ability of this test to identify S. equi direct from clinical sample material was not determined.
There is therefore a need to provide a robust and sensitive diagnostic test for horses infected with S. equi and in particular overcome the problems with conventional methodology primarily related to the existence of false-negative results.